The long-term goals of the proposed experiments are (i) to identify the DNA sequences required for replication origin function in mammalian cells and (ii) to identify the proteins which interact with replication origins to carry out initiation of DNA replication. First, replication origins will be localized with an accuracy of several hundred base pairs in several mammalian genomic regions using a two-dimensional (2D) gel electrophoretic method which we have already developed and tested with yeast cells. Second, the nucleotide sequences of the origin regions will be compared to identify possible common structural or sequence features. Third, the origin regions will be tested for their ability to allow circular DNA molecules containing them to replicate autonomously in mammalian cells. If origin-dependent autonomous replication can be rapidly tested by in vitro mutagenesis followed by autonomous replication assay. Fourth, DNA-protein interactions at origins in various cell cycle stages will be probed by in vivo "genomic footprinting" in order to identify sequences which interact with proteins. Fifth, proteins which interact with origin sequences will be identified and purified.